Biosensors are sensors that detect chemical species with high selectivity on the basis of molecular recognition rather than the physical properties of analytes. See, e.g., Advances in Biosensors, A.P.F. Turner, Ed. JAI Press, London, (1991). Many types of biosensing devices have been developed in recent years, including enzyme electrodes, optical immunosensors, ligand-receptor amperometers, and evanescent-wave probes. Updike and Hicks, Nature, 214: 986 (1967), Abdel-Latif et al., Anal. Lett., 21: 943 (111988); Giaever, J. Immunol., 110: 1424 (1973); Sugao et al. Anal. Chem., 65: 363 (1993), Rogers et al. Anal. Biochem., 182: 353 (1989).
Biosensors comprising a biological "binding molecule" attached to an optical fiber are well known in the prior art, most typically as evanescent wave detectors (see, for example, U.S. Pat. Nos. 4,447,546 to Hirschfeld and 4,582,809 and 4,909,990 to Block et al.). In order to maximize sensitivity and selectivity such biosensors typically utilize a single species of biological binding molecule affixed to the face of the sensor.
Such "single-species" biosensors are limited in that they have no inherent means to correct or calibrate for non-specific binding. Thus, they must be calibrated against an external standard. In addition, they are limited to the detection of a single analyte.
Biosensors comprising two or more species of biological binding partners overcome these limitations. A "multi-species" biosensor in principle permits simultaneous detection of as many different types of analytes as there are species of biological binding partner incorporated into the sensor. In addition, comparison of the amounts of a single analyte binding to multiple species of binding partner provides a measure of non-specific binding and thus acts as an intrinsic control for measurement variability introduced by non-specific binding.
In addition, the inclusion of fibers bearing biological binding partners specific for various analytes known to create a background signal in a particular assay provides a means for simultaneously measuring and substracting out the background signal. The provision of a multiplicity of fibers bearing different species of binding partner allows the detection of a multiplicity of moieties contributing to a background, or other, signal and the dissecting out of the contribution of each moiety to that signal.
To be most useful, a multi-species biosensor requires that the sensor provide a separate signal characterizing binding of analytes to each of the various species of binding partner comprising the probe. Thus each species of binding partner must be individually "addressed".
In addition, a "sensor face" (the surface bearing the biological binding partners) that has a relatively small surface area will facilitate measurement of small sample volumes as less sample material will be required to fully immerse the sensor face. A small surface area detector will also prove advantageous for use in in vitro measurements. Preparation of a detector bearing a large number of different biological binding partners that occupies a small area may be viewed as the preparation of a high density array of biological binding partners.
The creation of high density arrays of biological binding partners where each species of binding partner is uniquely addressed presents formidable fabrication problems. One of the most successful approaches, to date, is the large scale photolithographic solid phase synthesis pioneered by Affymax Inc. (see, e.g., Fodor et al. Science 251: 767-773 (1991) and U.S. Pat. No. 5,143,854). In this approach arrays of peptides or nucleic acids are chemically synthesized on a solid support. Different molecules are simultaneously synthesized at different predetermined locations on the substrate by the use of a photolithographic process that selectively removes photolabile protecting groups on the growing molecules in particular selected locations of the substrate. The resulting array of molecules is "spatially addressed". In other words the identity of each biological molecule is determined by its location on the substrate.
The photolithographic approach, however, is limited to molecules that can be chemically synthesized. Thus, it is typically restricted to peptides shorter than about 50 amino acids and nucleic acids shorter than about 150 base pairs. In addition, the photolithographic approach typically produces such arrays on a planar substrate (e.g. a glass slide) and provides no intrinsic mechanism by which a signal produced by the binding of a particular biological binding partner may be transmitted.
U.S. Pat. No. 5,250,264 to Walt et al. discloses a sensor comprising a fiber optic array using a "plurality of different dyes immobilized at individual spatial positions on the surface of the sensor." Each dye is capable of responding to a different analyte (e.g., pH, O.sub.2, CO.sub.2, etc.) and the sensor as a whole is capable of providing simultaneous measurements of multiple analytes.
Although the sensor disclosed by Walt et al. is not a biosensor, the reference describes a means of fabricating a sensor bearing a plurality of uniquely addressed "detection moieties". In Walt et al. optical fibers are first assembled to form a bundle. Transmission ends of a fiber or group of fibers of are then specifically illuminated. Each illuminated fiber transmits the light to its respective sensor end where the light "photopolymerizes" a sensor dye mixture causing the dye to bind to the sensor end. This process is repeated with different fibers for different photopolymerized dyes. This repetition continues until a sensor array is constructed.
This approach suffers from the limitations that it requires photopolymerizable sensor dyes and thus is limited in the number of different species per probe by the number of different dye type. In addition, this reference provides no means for attaching uniquely addressed biological molecules (e.g. peptides, nucleic acids, antibodies) to the sensor. Thus Walt et al. provide no means for the fabrication of biosensors.